During meiosis, high levels of recombination initiated by DNA double-strand breaks (DSBs) occur only after DNA replication. However, how DSB formation is coupled to DNA replication is unknown. We examined several DNA replication proteins for a role in this coupling in Schizosaccharomyces pombe, and we show that ribonucleotide reductase, the rate-limiting enzyme of deoxyribonucleotide synthesis and the target of the DNA synthesis inhibitor hydroxyurea (HU) is indirectly required for DSB formation linked to DNA replication. However, in cells in which the function of the DNA-replication-checkpoint proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, or Cds1p was compromised, DSB formation occurred at similar frequencies in the absence or presence of HU. The DSBs in the HU-treated mutant cells occurred at normal sites and were associated with recombination. In addition, Cdc2p is apparently not involved in this process. We propose that the sequence of meiotic S phase and initiation of recombination is coordinated by DNA-replication-checkpoint proteins.cell cycle ͉ DNA replication ͉ meiosis ͉ double-strand break formation D uring meiosis, DNA replication occurs, followed by two rounds of chromosome segregation (1, 2). A high level of homologous recombination occurs after meiotic DNA replication but before the first meiotic nuclear division (1, 3-7). Meiotic recombination is required for the proper segregation of homologs at meiosis I, because a chromatid of one homolog recombines with a chromatid of the other homolog, thereby joining homologous chromosomes.It is well established that meiotic recombination in both budding and fission yeasts is initiated by DNA double-strand breaks (DSBs), catalyzed by the DNA topoisomerase type-2-related enzyme Spo11 in budding yeast (8) and Rec12p in fission yeast (6). Both Spo11 in budding yeast and Rec12p in fission yeast are also essential for meiotic recombination. In addition to Rec12p, meiotic DSB requires multiple gene products that are also essential for meiotic recombination in two yeasts (1, 2).The DNA-replication checkpoint operates during both the mitotic cell cycle and meiosis to ensure that chromosome segregation is blocked if DNA replication is incomplete (9-12). The proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, and Cds1p are required for operation of the DNA-replication checkpoint in fission yeast (9-11, 13). The DNA-replicationcheckpoint proteins send a signal to block mitosis via the cyclin-dependent kinase Cdc2p complexed with the B-type cyclin Cdc13p (9). The Cdc2p-Cdc13p protein kinase is fully activated at the onset of mitosis by dephosphorylation of Cdc2p Tyr-15 (9). Phosphorylation of Cdc2p on Tyr-15 is catalyzed by both the Wee1p and Mik1p Tyr kinases, and dephosphorylation is carried out mainly by the Cdc25p Tyr phosphatase (14).It has been proposed that DNA replication is directly coupled to initiation of DSB formation in budding yeast, because inhibition of the completion of DNA replication either by hydroxyurea (HU) treatment or by mutation of both ...

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.